Marcos A. López-Patiño

The response of brain serotonergic and dopaminergic systems to an acute stressor in rainbow trout: a time course study

SUMMARY The brain monoaminergic neurotransmitter systems are known to be involved in the integrated response to stress in vertebrates. However, present knowledge about the timing of their actions as well as their specific roles in the regulation of the endocrine axes that drive the stress response is incomplete. This is partly because of the complexity of the reciprocal interactions among the monoaminergic systems and other biochemical effectors of the stress response such as corticotropin-releasing factor (CRF), arginine vasotocin (AVT), adrenocorticotropic hormone (ACTH) and corticosteroids. In this study, we show for the first time in teleost fish (rainbow trout) the short- and mid-term time course of the response of the forebrain serotonergic and dopaminergic activities after exposure to an acute stressor. Other stress markers like the plasma levels of cortisol, glucose and lactate were also monitored, providing a context in which to precisely locate the monoaminergic activation within the fish acute stress response. Our results show that acute stress induced a rapid increase in forebrain serotonergic activity, which became elevated after only 15 s of chasing. Several hours after stress, serotonergic activity recovered its basal levels, in parallel with the recovery of other stress markers such as plasma catecholamines and cortisol. Dopaminergic activity was also increased after stress, but only in the telencephalon and only after 20 min. The increase in serotonergic activity happened before the elevation of plasma catecholamines, suggesting that this monoamine system could have a key role in triggering the initial steps of the activation of not only the hypothalamus–pituitary–inter-renal axis but also the brain–sympathetic–chromaffin axis in fish.

Evidence of a metabolic fatty acid-sensing system in the hypothalamus and Brockmann bodies of rainbow trout: implications in food intake regulation

Enhanced lipid levels inhibit food intake in fish but no studies have characterized the possible mechanisms involved. We hypothesize that the presence of fatty acid (FA)-sensing mechanisms could be related to the control of food intake. Accordingly, we evaluated in the hypothalamus, hindbrain, and Brockmann bodies (BB) of rainbow trout changes in parameters related to fatty acid metabolism, transport of FA, nuclear receptors, and transcription factors involved in lipid metabolism, and components of the K(ATP) channel after intraperitoneal administration of different doses of oleic acid (long-chain fatty acid, LCFA) or octanoic acid (medium-chain fatty acid, MCFA). The increase in circulating LCFA or MCFA levels elicited an inhibition in food intake and induced in the hypothalamus a response compatible with fatty acid sensing in which fatty acid metabolism, binding to cluster of differentiation 36 (CD36), and mitochondrial activity are apparently involved, which is similar to that suggested in mammals except for the apparent capacity of rainbow trout to detect changes in MCFA levels. Changes in those hypothalamic pathways can be related to the control of food intake, since food intake was inhibited when FA metabolism was perturbed (using fatty acid synthase or acetyl-CoA carboxylase inhibitors) and changes in mRNA levels of specific neuropeptides such as neuropeptide Y and proopiomelancortin were also noticed. This response seems to be exclusive for the hypothalamus, since the other center controlling food intake (hindbrain) was unaffected by treatments. The results obtained in BB suggest that at least two of the components of a putative fatty acid-sensing system (based on fatty acid metabolism and binding to CD36) could be present. Therefore, the present study provides, for the first time in fish, evidence for a specific role for FA (MCFA and LCFA) as metabolic signals in hypothalamus and BB, where the detection of those FA can be associated with the control of food intake and hormone release.

Stress alters food intake and glucosensing response in hypothalamus, hindbrain, liver, and Brockmann bodies of rainbow trout

In fish food intake is altered under stress conditions, and in a fish teleost model like rainbow trout food intake is associated with the activity of the glucosensor systems. Thus, we aimed to evaluate the possible interaction of stress with the response of glucosensor mechanisms in rainbow trout. Thus, we subjected rainbow trout (via intraperitoneal injections) to normoglycaemic (control), hypoglycaemic (4 mg.kg(-1) bovine insulin) or hyperglycaemic (500 mg.kg(-1) glucose body mass) conditions for 5 days under normal stocking density (NSD, 10 kg fish mass·m(-3)) or stress conditions induced by high stocking density (HSD, 70 kg fish mass·m(-3)). The experimental design was appropriate since hypoglycemia and hyperglycemia were observed in fish under NSD whereas in normoglycaemic fish HSD induced changes in stress-related parameters similar to those reported in fish literature, such as increased levels of cortisol and glucose in plasma and decreased levels of glycogen in liver. Food intake did not respond to changes in plasma glucose levels in fish under HSD conditions, in contrast with the decreased food intake observed when glucose levels increased in fish under NSD conditions. Moreover, the changes with the increase in plasma glucose levels in parameters involved in glucosensing in liver, Brockmann bodies (BB), hypothalamus, and hindbrain of fish in NSD either disappeared (DHAP and GAP levels, and GK, PK, and GPase activities in liver; glucose, DHAP and GAP levels in BB; glucose and DHAP levels, and GK and PK activities in hypothalamus; glycogen and DHAP levels, and GSase activity in hindbrain) or changed (cortisol levels in plasma; glycogen and GAP levels, and GSase and FBPase activities in liver; GK and PK activities in BB; GK and PK activities in hindbrain) in fish under HSD. Those changes suggest for the first time in fish the existence of an interaction between glucosensing capacity and stress. The readjustment in the activity of glucosensor systems is also associated with changes in food intake resulting in an inability of the fish to compensate with changes in food intake those of circulating glucose levels as observed in fish under non-stressed conditions.

Oleic Acid and Octanoic Acid Sensing Capacity in Rainbow Trout Oncorhynchus mykiss Is Direct in Hypothalamus and Brockmann Bodies

In a previous study, we provided evidence for the presence in hypothalamus and Brockmann bodies (BB) of rainbow trout Oncorhynchus mykiss of sensing systems responding to changes in levels of oleic acid (long-chain fatty acid, LCFA) or octanoic acid (medium-chain fatty acid, MCFA). Since those effects could be attributed to an indirect effect, in the present study, we evaluated in vitro if hypothalamus and BB respond to changes in FA in a way similar to that observed in vivo. In a first set of experiments, we evaluated in hypothalamus and BB exposed to increased oleic acic or octanoic acid concentrations changes in parameters related to FA metabolism, FA transport, nuclear receptors and transcription factors, reactive oxygen species (ROS) effectors, components of the KATP channel, and (in hypothalamus) neuropeptides related to food intake. In a second set of experiments, we evaluated in hypothalamus the response of those parameters to oleic acid or octanoic acid in the presence of inhibitors of fatty acid sensing components. The responses observed in vitro in hypothalamus are comparable to those previously observed in vivo and specific inhibitors counteracted in many cases the effects of FA. These results support the capacity of rainbow trout hypothalamus to directly sense changes in MCFA or LCFA levels. In BB increased concentrations of oleic acid or octanoic acid induced changes that in general were comparable to those observed in hypothalamus supporting direct FA sensing in this tissue. However, those changes were not coincident with those observed in vivo allowing us to suggest that the FA sensing capacity of BB previously characterized in vivo is influenced by other neuroendocrine systems.

Central administration of oleate or octanoate activates hypothalamic fatty acid sensing and inhibits food intake in rainbow trout

If levels of fatty acids like oleate and octanoate are directly sensed through different fatty acid (FA) sensing systems in hypothalamus of rainbow trout, intracerebroventricular (ICV) administration of FA should elicit effects similar to those previously observed after intraperitoneal (IP) treatment. Accordingly, we observed after ICV treatment with oleate or octanoate decreased food intake accompanied in hypothalamus by reduced potential of lipogenesis and FA oxidation, and decreased potential of ATP-dependent inward rectifier potassium channel (K(+)ATP). Those changes support direct FA sensing through mechanisms related to FA metabolism and mitochondrial activity. The FA sensing through binding to FAT/CD36 and subsequent expression of transcription factors appears to be also direct but an interaction with peripheral hormones cannot be rejected. Moreover, decreased expression of NPY and increased expression of POMC were observed in parallel with the activation of FA sensing systems and decreased food intake. These results allow us to suggest the involvement of at least these peptides in controlling the decreased food intake noted after oleate and octanoate treatment in rainbow trout.

Short-term time course of liver metabolic response to acute handling stress in rainbow trout, Oncorhynchus mykiss.

To elucidate the short-term time-course of liver metabolic response in rainbow trout to acute handling stress we subjected rainbow trout to 5min chasing and obtained samples 0 to 480min post-stress. Levels of cortisol, glucose and lactate were measured in plasma, whereas metabolite levels, enzyme activities, mRNA abundance of parameters related to energy metabolism, and glucocorticoid receptors were assessed in liver. Acute stress affected many parameters related to energy metabolism, with most of them turning back to normal levels after 480min. In general, the present results support the existence of two stages in the short-term time-course of metabolic response to handling stress. A first stage occurring few minutes post-stress (15-45min), was characterized by increased mobilization of liver glycogen resulting in increased production of endogenous glucose, reduced use of exogenous glucose and reduced lipogenic potential. A second stage, occurring 60-120min post-stress onwards was characterized by the recovery of liver glycogen levels, the increased capacity of liver for releasing glucose, and the recovery of lipogenic capacity whereas no changes were noted in gluconeogenic potential, which probably needs longer time periods to become enhanced.

Ghrelin modulates hypothalamic fatty acid-sensing and control of food intake in rainbow trout.

There is no information available on fish as far as the possible effects of ghrelin on hypothalamic fatty acid metabolism and the response of fatty acid-sensing systems, which are involved in the control of food intake. Therefore, we assessed in rainbow trout the response of food intake, hypothalamic fatty acid-sensing mechanisms and expression of neuropeptides involved in the control of food intake to the central treatment of ghrelin in the presence or absence of a long-chain fatty acid such as oleate. We observed that the orexigenic actions of ghrelin in rainbow trout are associated with changes in fatty acid metabolism in the hypothalamus and an inhibition of fatty acid-sensing mechanisms, which ultimately lead to changes in the expression of anorexigenic and orexigenic peptides resulting in increased orexigenic potential and food intake. Moreover, the response to increased levels of oleate of hypothalamic fatty acid-sensing systems (activation), expression of neuropeptides (enhanced anorexigenic potential) and food intake (decrease) were counteracted by the simultaneous treatment with ghrelin. These changes provide evidence for the first time in fish of a possible modulatory role of ghrelin on the metabolic regulation by fatty acid of food intake occurring in the hypothalamus.

Evidence for the Presence of Glucosensor Mechanisms Not Dependent on Glucokinase in Hypothalamus and Hindbrain of Rainbow Trout (Oncorhynchus mykiss).

We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) operate in hypothalamus and hindbrain of the carnivorous fish species rainbow trout and stress affected them. Therefore, we evaluated in these areas changes in parameters which could be related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and sodium/glucose co-transporter 1 (SGLT-1) 6h after intraperitoneal injection of 5 mL.Kg-1 of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment, 4 mg bovine insulin.Kg-1 body mass), or D-glucose (hyperglycaemic treatment, 500 mg.Kg-1 body mass). Half of tanks were kept at a 10 Kg fish mass.m-3 and denoted as fish under normal stocking density (NSD) whereas the remaining tanks were kept at a stressful high stocking density (70 kg fish mass.m-3) denoted as HSD. The results obtained in non-stressed rainbow trout provide evidence, for the first time in fish, that manipulation of glucose levels induce changes in parameters which could be related to putative glucosensor systems based on LXR, mitochondrial activity and sweet taste receptor in hypothalamus, and a system based on SGLT-1 in hindbrain. Stress altered the response of parameters related to these systems to changes in glycaemia.

ACTH-stimulated cortisol release from head kidney of rainbow trout is modulated by glucose concentration

SUMMARY To assess the hypothesis that cortisol release in rainbow trout is modulated by glucose levels, we first evaluated cortisol release [basal and adrenocorticotropic hormone (ACTH)-regulated] by head kidney tissue superfused with medium reflecting hypoglycaemic, normoglycaemic or hyperglycaemic conditions. Next, cortisol release from head kidney fragments in static incubations was assessed in parallel with changes in parameters related to cortisol synthesis (mRNA abundance of StAR, P450scc, 3βHSD and 11βH) and the GK-mediated glucosensing mechanism (levels of glycogen and glucose, activities of GK, GSase and PK, and mRNA levels of GK, GLUT-2, Kir6.x-like and SUR-like). We then evaluated the effects of two inhibitors of glucose transport, cytochalasin B and phlorizin, on cortisol production and glucosensing mechanisms. The ACTH-induced release of cortisol proved to be modulated by glucose concentration such that increased release occurs under high glucose levels, and decreased ACTH-stimulated cortisol release occurs when glucose transport is inhibited by cytochalasin B. The release of cortisol can be associated with increased synthesis as enhanced mRNA abundance of genes related to cortisol synthesis was also noted in high glucose medium. Specific GK immunoreactivity in the cortisol-producing cells (not in chromaffin cells) further substantiates GK-mediated glucosensing in cortisol production. In contrast, no changes compatible with those of glucose levels and cortisol release/synthesis in the presence of ACTH were noted for any other putative glucosensor mechanisms based on LXR, SGLT-1 or Gnat3. These combined results are the first evidence for a mechanism in fish linking the synthesis and release of a non-pancreatic hormone like cortisol with circulating glucose levels. The relationship was evident for the regulated (ACTH-dependent) pathway and this suggests that under acute stress conditions glucose is important for the regulation of cortisol synthesis and release.

Counter-regulatory response to a fall in circulating fatty acid levels in rainbow trout. Possible involvement of the hypothalamus-pituitary-interrenal axis.

We hypothesize that a decrease in circulating levels of fatty acid (FA) in rainbow trout Oncorhynchus mykiss would result in the inhibition of putative hypothalamic FA sensing systems with concomitant changes in the expression of orexigenic and anorexigenic factors ultimately leading to a stimulation of food intake. To assess this hypothesis, we lowered circulating FA levels treating fish with SDZ WAG 994 (SDZ), a selective A1 adenosine receptor agonist that inhibits lipolysis. In additional groups, we also evaluated if the presence of intralipid was able to counteract changes induced by SDZ treatment, and the possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis by treating fish with SDZ in the presence of metyrapone, which decreases cortisol synthesis in fish. The decrease in circulating levels of FA in rainbow trout induced a clear increase in food intake that was associated with the decrease of the anorexigenic potential in hypothalamus (decreased POMC-A1 and CART mRNA abundance), and with changes in several parameters related to putative FA-sensing mechanisms in hypothalamus. Intralipid treatment counteracted these changes. SDZ treatment also induced increased cortisol levels and the activation of different components of the HPI axis whereas these changes disappeared in the presence of intralipid or metyrapone. These results suggest that the HPI axis is involved in a counter-regulatory response in rainbow trout to restore FA levels in plasma.